Sexual transmission is the leading cause of new HIV-1 infections worldwide. There is increasing evidence that HSV-2 is an important cofactor for HIV-1, likely due to genital inflammation associated with HSV-2 infection. Co-infected individuals are also more likely to transmit HIV-1, and increased HIV-1 shedding into the genital tract has been observed. Up to 90 percent of people with HIV-1 are infected with HSV-2, and treatment with acyclovir reduces HIV-1 genital secretions, and thereby HIV-1 transmission. Therefore, preventing HSV-2 should have the added benefit of reducing HIV-1 transmission. However, currently there are no vaccines or other preventative strategies for HSV-2. A major hurdle for rational vaccine design is the lack of knowledge of the correlates for HSV-2 protection. Innate immune responses, through pathogen-mediated ligation of pattern recognition receptors (PRRs), such as the toll-like receptors (TLRs), are required for immune responses to HSV-2. However, there is little mechanistic insight regarding TLR-mediated regulation of HSV-2 responses. TLRs are well represented on both hematopoietic and somatic cell types. Combined with the heterogeneity of dendritic cells (DCs), the major cell type for initiating immune responses, use of standard knockout mice to delineate TLR mechanism of action is challenging. RNA interference (RNAi) is a mechanism of post-transcriptional gene silencing. Previously, we have used small interfering (si)RNAs to protect mice from vaginal HSV-2 transmission. We propose to develop an RNAi- based method to study the effects of DC-localized TLRs in vivo. We will use antibody (Ab)-based targeting to deliver siRNAs, specific for single and combinations of TLRs to different DC subsets. We will characterize these reagents in vitro and use them to assess the role of specific sets of TLRs in a mouse model of HSV-2. By deciphering the contribution of TLRs individually and in combination on different DC subsets in HSV-2 infection, we can begin to visualize which DC-localized TLRs are critical for optimal immune responses. With the development of TLR agonists as immune stimulators to prevent HSV-2 transmission, these studies will further our understanding of which components of TLR-stimulated immunity are required for immune activation. PUBLIC HEALTH RELEVANCE: HSV-2 is a major co-factor for HIV-1, likely due to the inflammatory responses associated with infection. Preventing HSV-2 transmission should have a major impact on reducing HIV-1 infections. A major hurdle for designing preventative therapies is a lack of knowledge of the correlates of HSV-2 protection. How pattern recognition receptors trigger immune responses is critical in furthering our understanding of pathogenic infections. We will assess the role of toll-like receptors, localized on dendritic cells to better define these interactions in an HSV-2 infection model.